Arrestin interactions with non-receptor binding partners

描述（由申请人提供）：抑制蛋白首先被描述为通过G蛋白的G蛋白偶联受体（GPCR）信号传导的负调节剂。新的数据表明，游离和受体结合的抑制蛋白通过MAP激酶启动信号传导，MAP激酶调节细胞死亡、存活和增殖。特别是，游离的和受体相关的arrestin-3支架 ASK 1-MKK-JNK级联，促进JNK活化。在这里，我们建议阐明抑制蛋白依赖性激活促凋亡JNK家族激酶及其激活剂MKK 4/7的结构基础，使用生物化学和生物物理学方法。将建立由arrestin-3组织的多蛋白信号传导复合物（信号体）的组装的分子机制，并且将鉴定对JNK活化至关重要的arrestin-3残基。在此基础上，构建了结合ASK 1、MKK和JNK，但不促进JNK活化的arrestin-3突变体。将测试具有显著降低的激活JNK的能力的抑制蛋白突变体的潜力，其中一些我们已经有了，以保护细胞免受损伤并延长其存活。我们还构建了抑制蛋白-3突变体，其比亲本野生型抑制蛋白-3更有效地激活JNK。我们建立了arrestin-3依赖性JNK激活在细胞存活中起关键作用的范例。我们将测试过度活跃的arrestin-3突变体促进细胞死亡的能力。我们发现失活突变体将JNK和上游激酶捆绑在非生产性复合物中，从而以显性负性方式起作用。我们将测试这些突变体保护细胞并延长其存活的潜力。特异性增加或阻断促凋亡信号传导的分子工具在疾病中具有治疗潜力 与过度的细胞增殖有关（例如，癌症）或死亡（例如，神经变性疾病）。 公共卫生相关性：该提议集中于使用生物化学和生物物理方法阐明促凋亡JNK家族激酶及其激活剂MKK 4/7的抑制蛋白依赖性激活的结构基础。基于此信息构建的抑制蛋白突变体激活JNK的能力显著降低， 将测试细胞抵抗损伤并延长其存活的能力，以及还将测试比野生型抑制蛋白更有效地激活JNK以促进细胞死亡的突变体的能力。特异性增加或阻断促凋亡信号传导的分子工具在与过度细胞增殖相关的病症（例如，癌症）或死亡（例如，神经变性疾病）。

DESCRIPTION (provided by applicant): Arrestins were first described as negative regulators of G protein-coupled receptor (GPCR) signaling via G proteins. New data show that the free and receptor-bound arrestins initiate signaling through MAP kinases, which regulate cell death, survival, and proliferation. In particular, both free and receptor- associated arrestin-3 scaffolds ASK1-MKK-JNK cascade, promoting JNK activation. Here we propose to elucidate the structural basis of arrestin-dependent activation of pro-apoptotic JNK family kinases and their activators MKK4/7 using biochemical and biophysical methods. The molecular mechanisms of the assembly of multi-protein signaling complexes (signalosomes) organized by arrestin-3 will be established, and arrestin-3 residues critical for JNK activation will be identified. Based on ths info, arrestin-3 mutants that bind ASK1, MKK and JNK, but do not promote JNK activation will be constructed. The potential of arrestin mutants with dramatically reduced ability to activate JNKs, several of which we already have, to protect cells against insults and prolong their survival will be tested. We have also constructed arrestin-3 mutants that activate JNKs more efficiently than parental wild type arrestin-3. We established the paradigm where arrestin-3-dependent JNK activation plays key role in cell survival. We will test the ability of hyperactive arrestin-3 mutants to facilitate cell death. We showed that inactive mutants tie up JNKs and upstream kinases in unproductive complexes, thereby acting in dominant- negative manner. We will test the potential of these mutants to protect cells and prolong their survival. Molecular toos that specifically increase or block pro-apoptotic signaling have therapeutic potential in disorders associated with excessive cell proliferation (e.g., cancer) or death (e.g., neurodegenerative diseases). PUBLIC HEALTH RELEVANCE: This proposal focuses on the elucidation of the structural basis of arrestin-dependent activation of pro- apoptotic JNK family kinases and their activators MKK4/7 using biochemical and biophysical methods. The potential of arrestin mutants with dramatically reduced ability to activate JNKs, that were constructed based on this info, to protect cells against insults and prolong their survival will be tested, as well as the ability of the mutats that activate JNKs more efficiently than wild type arrestins to facilitate cell death will be also tested. Molecular tools that specifically increase or block pro-apoptotic signaling have therapeutic potential in disorders associated with excessive cell proliferation (e.g., cancer) or death (e.g., neurodegenerative diseases).